Human H/ACA ribonucleoproteins (RNPs) are important for many basic cellular processes including protein synthesis, pre-mRNA splicing, and genome integrity. Among a growing number of functions, H/ACA RNPs isomerize some 130 uridines to pseudouridines in ribosomal (r) and spliceosomal small nuclear (sn) RNAs, process rRNA, stabilize telomerase RNA, and yield microRNAs. Each of these functions is specified by one of over 150 unique H/ACA RNAs, each of which associates with the same four core proteins to form an H/ACA RNP. The central core protein NAP57 (aka dyskerin) is mutated in the predominant X-linked form of the inherited bone marrow failure syndrome dyskeratosis congenita. Although consisting of only five components, biogenesis of these particles is surprisingly complex requiring at least four assembly factors, SHQ1, NAF1, and pontin and reptin. Our recent demonstration that dyskeratosis congenita mutations in NAP57 modulate its interaction with SHQ1 marks DC as an RNP assembly deficiency. This proposal will elucidate mechanisms of assembly factor function in H/ACA RNP biogenesis and thereby not only advance the field but also inform on the basic mechanism of a human disease. This will be approached in the following four Aims: by defining (i) the function of SHQ1 vis-`-vis NAP57, (ii) the role of pontin and reptin in SHQ1 removal from NAP57, (iii) the function of NAF1 in H/ACA RNP biogenesis, and (iv) by identifying the full complement for H/ACA RNP assembly factors. To achieve these goals, we will develop novel approaches, such as dual-color fluorescence fluctuation spectroscopy for the study of protein-protein interaction in living cells, and rely on our established assay systems, such as in vitro assembly of functional H/ACA RNPs in cytosolic extracts and in vivo assembly in our H/ACA RNA inducible cell line. These studies are intended to work towards our long-term goals to define all H/ACA RNP assembly factors for in vitro reconstitution of functional RNPs from recombinant components, to characterize the molecular basis of dyskeratosis congenita by comparing wild type and mutant particles, to obtain high resolution structures of H/ACA RNPs, and to spatially define the process of H/ACA RNP biogenesis relative to subcellular location. PUBLIC HEALTH RELEVANCE: In general, this proposal will shed light on the assembly mechanism of small nucleolar ribonucleoproteins, which are important for such basic cellular processes as ribosome biogenesis and function, pre-mRNA splicing, genome integrity, and protein translation. Therefore, the proposed studies will promote our general understanding of cell physiology, which serves as basis for the treatment of many diseases. In particular, our studies are intended to shed light on the mechanism of the X-linked bone marrow failure syndrome dyskeratosis congenita and can eventually be exploited for the identification of drugs that will be beneficial to patients with dyskeratosis congenita.